Combined cytogenetic, somatic cell genetic, immunological and molecular methods will be used to clarify the role of gene amplification in carcinogenesis and tumor progression. Attention will be focused on mammalian ribosomal RNA genes, in which amplification is associated with hypermethylation and transcriptional inactivity. We will attempt to activate them by demethylation with 5-azacytidine in order to establish if DNA methylation has a role in rRNA gene regulation. Restriction mapping of the rDNA of inactive and reactivated genes may identify any sites of methylation involved in rRNA gene regulation. The relationship between amplification and hypermethylation of rRNA genes will be studied in various ways, e.g., by amplifying active, nonmethylated genes by co-transformation with cloned rRNA and DHFR sequences. The nature of the amplified sequences in untreated tumor cells will be studied using cloned fragments of the amplified sequences.